SummaryThe ATR and ATM protein kinases are known to be involved in a wide variety of responses to DNA damage. The Arabidopsis thaliana genome includes both ATR and ATM orthologs, and plants with null alleles of these genes are viable. Arabidopsis atr and atm mutants display hypersensitivity to c-irradiation. To further characterize the roles of ATM and ATR in response to ionizing radiation, we performed a short-term global transcription analysis in wild-type and mutant lines. We found that hundreds of genes are upregulated in response to cirradiation, and that the induction of virtually all of these genes is dependent on ATM, but not ATR. The transcript of CYCB1;1 is unique among the cyclin transcripts in being rapidly and powerfully upregulated in response to ionizing radiation, while other G 2 -associated transcripts are suppressed. We found that both ATM and ATR contribute to the induction of a CYCB1;1:GUS fusion by IR, but only ATR is required for the persistence of this response. We propose that this upregulation of CYCB1;1 does not reflect the accumulation of cells in G 2 , but instead reflects a still unknown role for this cyclin in DNA damage response.

Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atrÿ/ÿ mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to g-radiation. G2 arrest was observed in response to g-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (␥-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of ␥ -H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, ϳ10% of IR-induced ␥-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, ␥-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals. INTRODUCTIONThe induction of DNA double-strand breaks (DSBs) in eukaryotes triggers a number of protective responses including the upregulation of repair pathways, initiation of cell cycle arrest, and, in some organisms, the induction of programmed cell death. DSBs in actively dividing cells are particularly dangerous. Repair to form translocations and deletions can lead to loss of heterozygosity, which in turn leads to carcinogenesis in mammals or lethality in haploid yeast. For this reason all living things possess the ability to detect the presence of DSBs and relay this information to the cell cycle.Two important protein kinases involved in sensing and signaling DNA damage in eukaryotes are ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and rad3-related (ATR; Abraham, 2001;Sancar et al., 2004). In mammals, ATM is critical for responses to DSBs and signals downstream cell cycle checkpoint regulators including p53 and Chk2 to coordinate apoptotic responses and/or cell cycle arrest (Fernandez-Capetillo et al., 2002). In addition to checkpoint regulation, ATM responds to DSBs by interacting with proteins intimately involved in DNA repair such as the Mre11-Rad50-Nbs1 (M-R-N) complex (Gatei et al., 2000;van den Bosch et al., 2003) and RAD51 (Chen et al., 1999). In comparison, ATR, in a complex with the ATR interacting protein (ATRIP), is thought to respond primarily to agents that block replication, recognizing stalled replication forks and then signaling to Chk1 and p53 to induce cell cycle arrest, replication restart, and apoptosis (Abraham, 2001). In striking contrast to ATM, ATR is an essential gene in mammals; defects in the murine homolog cause early embryonic lethality and loss of ATR in conditional knock-out embryonic stem cells rapidly...

Light plays a key role in the development and physiology of plants. One of the most profound effects of light on plant development is the derepression of expression of an array of light-responsive genes, including the genes encoding the chlorophyll alb binding proteins (CAB) of photosystem II. To understand the mechanism by which light signals nuclear gene expression, we developed a genetic selection to identify mutants with reduced CAB transcription. Here, we describe a new Arabidopsis locus, COE7 (for CAS ynderexpressed). Mutations at this locus result in defects in expression of several light-regulated genes, specifically in mesophyll but not in bundle-associated or epidermis cells. Reduced accumulation of CAB and other photosynthesis-related mRNAs in the mesophyll was correlated with defects in chloroplast development in these cells, resulting in a reticulate pattern with veins greener than the interveinal regions of leaves.Moreover, chalcone synthase mRNA, although known to be regulated by both phytochrome and a blue light receptor, accumulated normally in the leaf epidermis. Dark basal levels of CAB expression were unaffected in etiolated cueí seedlings; however, induction of CAB transcription by pulses of red and blue light was reduced, suggesting that CUEl acts downstream from both phytochrome and blue light photoreceptors. CUEl appears to play a role in the primary derepression of mesophyll-specific gene expression in response to light, because cueí mutants are severely deficient at establishing photoautotrophic growth. Based on this characterization, we propose that CUEí is a cell-specific positive regulator linking light and intrinsic developmental programs in Arabidopsis leaf mesophyll cells.